Fourteen Manitoba legume seeds and a pea protein isolate were evaluated for ferritin production. Optimized ferritin concentrate yields from 5 selected isolates ranged from 19.07% (Chick Peas) to 9.69% (Moon Dal Washed). Iron concentrations were between 0.45g (Green Lentils Whole) and 0.30g (Chick Pea)/100g. SDS-PAGE revealed presence of the major ferritin polypeptides in the concentrates. The levels of iron in ferritin appear to be directly related to the amount of negatively charged amino acids. Intrinsic fluorescence and circular dichroic spectra showed that the ferritin concentrates had denatured protein conformations at pH 2 and 7, which is critical to their digestibility and bioavailability. Gel-permeation chromatography revealed differences in elution volumes between pre- and post-digestion ferritin concentrates, and kinetics studies confirmed susceptibility to proteolysis and a high potential for iron release. Results demonstrated the feasibility of phytoferritin production from Manitoba pulses, which could serve as a better iron supplement than inorganic iron during IDA treatment. / October 2016

Three groups of goldfish were prepared; naive, avoidance-conditioned and pseudo-conditioned animals. Five pseudo-conditioned fish were avoidance trained later and found to have no measurable acquisition of the avoidance conditioning paradigm. Several protein fractions were found to have significantly different rates of synthesis when compared across groups. The possible involvement of these proteins in the memory storage process was discussed.

No description available.

Call number: LD2668 .T4 1952 B8 / Master of Science

Computational methods of analyzing, simulating, and modeling proteins are essential towards understanding protein structure and its interactions. Computational methods are easier as not all protein structures can be determined experimentally due to the inherent difficultly of working with some proteins. In order to predict, design, analyze, simulate or model a protein, data from experimentally determined proteins such as those located in the repository of the Protein Data Bank (PDB) are essential. The assumption here is that we can use pieces of known proteins to piece together a "new" protein hence, de novo protein design. The analysis of the geometric relationships between secondary structure elements in proteins can be extremely useful to protein prediction, analysis, and de novo design. This thesis project involves creating a database of protein secondary structure elements and geometric information for rapid protein assembly, de novo protein design, prediction and analysis.

Die Herzinsuffizienz ist eine der führenden Todesursachen weltweit. Eine auf die neurohumorale Aktivierung zugeschnittene Therapie mit ACE-Hemmern bzw. Angiotensin-Rezeptorantagonisten, Betablockern, Aldosteronantagonisten und Diuretika verbessert zwar die Symptomatik und Prognose. Letztere ist bei Diagnosestellung jedoch immer noch schlechter als die vieler maligner Erkrankungen einzuschätzen. Ziel ist daher die Entwicklung von Medikamenten, die den Krankheitsverlauf des Syndroms Herzinsuffizienz aufhalten bzw. umkehren. Ein Ansatz ist dabei die Analyse sogenannter Kandidatengene, die im kranken Herzen differentiell exprimiert werden und potentiell medikamentös beeinflussbar sind. Im Rahmen der vorliegenden Arbeit wurden zwei solcher Kandidatengene charakterisiert. Mäuse mit kardialer Überexpression b1-adrenerger Rezeptoren entwickeln eine Herzinsuffizienz mit kardialer Hypertrophie und Fibrose. In Gen Arrays mit 21000 Maus-ESTs zeigten sich unter anderem die Gene des Uncoupling Protein 2 (UCP2) und der Diphosphoinositol-Polyphosphat-Phosphohydrolase 2 alpha (DIPP2a) aktiviert. Diese Befunde wurden zunächst mittels RNase Protection Assay und RT-PCR bestätigt. Auch andere murine Herzinsuffizienzmodelle wurden untersucht. So ließ sich ebenfalls im druckinduzierten Herzinsuffizienzmodell nach artifizieller Aortenstenose sowie im b2-AR überexprimierenden Herzen eine erhöhte Konzentration von UCP2- und DIPP2a-mRNA messen. Um zu prüfen, ob diese differentielle mRNA-Expression deletäre oder protektive Effekte vermittelt, wurden jeweils transgene Mauslinien mit herzspezifischer Überexpression von UCP2 und DIPP2a generiert. Die Linie UCP2-TG1 mit hoher Überexpression sowie ein Gründer-Tier der UCP2-transgenen Mäuse entwickelten eine Herzinsuffizienz mit vergrößertem Herzen, linksventrikulärem Pumpversagen, interstitieller Fibrose und typischen Veränderungen der molekularen Marker ANF und SERCA. Zudem fanden sich dilatierte Vorhöfe sowie eine bradykarde Herzrhythmusstörung. UCP2 ist ein Entkoppler der oxidativen Phosphorylierung im Mitochondrium. Im energieintensiven Stoffwechsel des Myokards könnte eine durch UCP2 reduzierte ATP-Synthese zu den genannten Veränderungen führen. Für UCP2 wurden auch protektive Eigenschaften durch das Abfangen freier Radikale beschrieben. Die Linie UCP2-TG3 mit niedrigerem Überexpressionsniveau und ohne kardialen Phänotyp wurde deshalb einem Aortic banding unterzogen, wo sich in der Überlebenskurve kein protektiver oder deletärer Effekt einer moderat vermehrten Entkopplung im Vergleich zum Wildtyp zeigte. In drei unabhängigen Linien transgener Mäuse mit herzspezifischer Überexpression von DIPP2a ließ sich morphometrisch eine kardiale Hypertrophie nachweisen. Die Linie DIPP2a-TG9 mit dem höchsten Überexpressionsniveau zeigte zudem eine kardiale Fibrose sowie unter Dobutamin eine verminderte kardiale Kontraktilitätsreserve im Vergleich zum Wildtypen. DIPP-Proteine hydrolysieren Inositolphosphate und Nukleosiddiphosphate und greifen so in zentrale Stoffwechselvorgänge ein, die im einzelnen noch nicht geklärt sind. Es konnte hier im Mausmodell gezeigt werden, dass UCP2 und DIPP2a zwei für die Entwicklung einer Herzinsuffizienz relevante Zielproteine darstellen. Geplant ist die weitere Aufklärung der beteiligten Mechanismen, um diese letztlich auch therapeutisch beeinflussen zu können. / The expression of uncoupling protein 2 (UCP2) and diphosphoinositol polyphosphate phosphohydrolase 2 alpha (DIPP2a) was shown to be upregulated in a murine model of chronic heart failure overexpressing beta1-adrenergic receptors and in other heart failure models. These candidate genes could play a protective or detrimental role in the development of chronic cardiac insufficiency. To test this hypothesis mice overexpressing UCP2 and DIPP2a, respectively, were generated. UCP2-transgenes with high expression level developed a chronic heart failure with interstitial fibrosis, cardiac hypertrophy and altered molecular markers of heart failure. They showed dysplastic atria and a bradyarrhythmia. DIPP2a-transgens with high expression level showed minor fibrotic changes in the myocardium in advanced age. Cardiac reserve was reduced as well. UCP2 and DIPP2a are two candidate genes which might be involved in the development of chronic heart failure.

Thesis (Sc.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The c-Jun NH2-terminal kinase (JNK) and p38 intracellular signaling cascades are mitogen-activated protein kinase (MAPK) signaling pathways that are activated in mammalian cells by a variety of stressors, including proinflammatory cytokines, osmotic shock, and shear stress. The purpose of this dissertation research was to examine the effect of injury-producing exercise on JNK and p38 activities in human skeletal muscle and to determine whether mechanical stress is a primary stimulator of JNK and p38 activities with contraction. Twelve healthy subjects (7M/5F) completed maximal concentric or eccentric knee extensions on an isokinetic dynamometer (10 sets, 10 reps). Needle biopsies were obtained from the vastus lateralis muscle 24 h before exercise, immediately post-exercise, and 6 h post-exercise. While both forms of exercise increased JNK activity immediately post-exercise, eccentric contractions resulted in a much higher activation (15-fold vs. 4-fold increase above basal for eccentric and concentric, respectively). By 6 h post-exercise, JNK activity decreased back to baseline values. In a separate study, 14 male subjects completed a 42.2 km marathon. Biopsies were obtained from the vastus lateralis muscle 10 days prior to the marathon, immediately following the race, and 1, 3, and 5 days after the race. JNK activity increased 7-fold over basal immediately postexercise, but decreased back to basal 1, 3, and 5 days after the exercise. The activity of p38y also was increased and decreased in a similar pattern. However, no regulation was observed for p38α. In a third study, the effects of contraction and static stretch on JNK activity and p38 phosphorylation were determined in the rat soleus muscle in vitro. Static stretch dramatically increased JNK activity and p38 phosphorylation, whereas isometric contraction resulted in much smaller increases in JNK activity and p38 phosphorylation. The regulation of focal adhesion proteins also was examined following both exercise and contraction. The work presented in this thesis demonstrates that injury-producing exercise results in the marked activation of the JNK and p38 stress-activated protein kinases and provides evidence that mechanical stress may be a major contributor to increases in JNK and p38 activities observed following contraction in rat and human skeletal muscle. / 2031-01-01

A dissertation submitted to the Faculty of Science, University of Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014.

A research report Submitted to the School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Masters of Science in Epidemiology and Biostatistics 15 June 2017, Johannesburg, South Africa / Introduction: Worldwide, cardiovascular diseases are the number one cause of death with a three-quarter of cardiovascular disease deaths occurring in low-middle income countries. Childhood social adversity as a proxy of psychosocial stress has been found to be associated with later adult risk of cardiovascular diseases, with studies investigating the mechanisms linking early exposure to social adversity and later risk of cardiovascular diseases. CRP has been a biomarker that is found to be associated with the risk of cardiovascular diseases in adults, however, the association between CRP levels in adolescence and social adversity in children (prenatal and postnatal periods) is not well documented. Assessing the association between childhood social adversity and CRP levels in late adolescent period will encourage further studies to explore whether high levels of CRP tracks from adolescence to adulthood and ultimately increase the risk of cardiovascular diseases in the South African context. Aim: This study aims to determine the association between social adversity from the prenatal period to two years of age and the level of CRP in the same cohort at the age of 18 (from 1990 to 2008) Methods: The study was a secondary data analysis of the Birth to Twenty longitudinal study which recruited 3273 singleton children. Four measures (prenatal and postnatal (0-2 years)) of social adversity in children (which are maternal prenatal stress, maternal prenatal general feeling, maternal postnatal depression and household socioeconomic status) were used. The high-sensitivity C-reactive protein was grouped into tertiles (1st tertile: hs-CRP<0.48 mg/l, 2nd tertile: 0.48<hs-CRP<1.16, 3rd tertile: hs-CRP>1.16) and multinomial logistic regressions were therefore used to assess the association between childhood social adversities and tertiles of high sensitivity C-reactive protein. Results: The primary Birth to Twenty longitudinal study had more than 35% loss to follow-up at 18 years. No statistically significant difference (p>0.05) was found on demographic variables of those included in the analysis compared to those not included (due to current study criteria and loss to follow-up). A unit increase in maternal marital stress score during pregnancy was associated with an increase by 2.23 (p=0.03) in the relative risk of the youth being in the 2nd high sensitivity C-reactive protein tertile in comparison to being in the 1st high sensitivity C-reactive protein tertile. For a unit increase in maternal family stress score, the relative risk of the youth being in the 3rd high sensitivity C-reactive protein is 1.61 (p=0.04) times greater in comparison to being in the 1st high sensitivity C-reactive protein tertile. No statistically significant associations were observed among the other categories of social adversity (p>0.05) and high sensitivity C-reactive proteins. Low social support to mothers during pregnancy was associated with elevated high-sensitivity C-reactive protein in adolescents. Conclusion: A positive association was observed between a prenatal measure of social adversity and elevated high-sensitivity C-reactive protein; In particular, increased levels of family and relationship-related prenatal stress during pregnancy is a predictor of elevated high-sensitivity C-reactive protein in children. This study contributes to the empirical evidence from studies done in animals suggesting that early development of adult health complication starts during the intrauterine period. The findings of this study will further guide intervention research to target conditions during intrauterine period in preventing adult health complications. / MT2017

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The c-Jun N-terminal kinases (JNKs) are mitogen-activated protein kinases (MAPKs) that are activated by the dual phosphorylation of a canonical threonine and tyrosine residue. While it is well known that the activation of JNK mediates many important cellular processes such as differentiation, proliferation, and apoptosis, the mechanisms by which phosphorylation induces its activation are not known. An understanding of the structural and biophysical basis for the activation of JNK is highly desirable however, as dysregulation of the kinase has been implicated in numerous prominent diseases. Aiming first to improve upon the previously reported inadequacies in acquiring active JNK, this work describes a novel method for the purification of large yields of pure and phosphorylated JNK1β1, the most abundant JNK isoform. Using codon harmonization as a precautionary measure toward increasing the soluble overexpression of the kinase raised unique questions about the role of translation kinetics in both the heterologous and natural co-translational modification of kinases. After purifying the upstream activating kinases of JNK, phosphorylation of JNK1β1 was achieved by reconstituting the MEKK1 → MKK4 → JNK MAPK activation cascade in vitro. Activated JNK1β1 was thereafter able to phosphorylate its substrate, ATF2, with high catalytic efficiency. Characterising the nature of JNK1β1 modification by MKK4, mass spectrometry revealed that the latter kinase phosphorylates JNK1β1 not only at its activation residues (T183 and Y185), but also at a recognised yet uncharacterised phospho-site (S377) as well as two novel phospho-residues (T228 and S284) whose phosphorylation appear to have functional significance. Unfolding studies and amide hydrogen-deuterium exchange (HX) mass spectrometry (MS) were then used to investigate the changes to the stability and structure/conformational dynamics of JNK1β1 induced by phosphorylation and nucleotide substrate binding. Increased flexibility detected at the hinge between the N- and C-terminal domains upon phosphorylation suggested that activation may require interdomain closure. Patterns of solvent protection by the ATP analogue, AMP-PNP, reflected a novel mode of nucleotide binding to the C-terminal domain of a destabilised and open domain conformation of inactive JNK1β1. HX protection at both domains following AMP-PNP binding to active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, simultaneously stabilising the kinase. This reveals that phosphorylation activates JNK1β1 in part by enhancing the flexibility of the hinge to enable interdomain closure and the formation of a functional active site. This work thus offers novel insight into the unique molecular mechanisms by which JNK1β1 is regulated by nucleotide binding and phosphorylation by MKK4, and by the complex interplay that exists between them.